A number of laboratories have provided data suggesting that nondividing cells contain intracellular proteins that actively inhibit cell proliferation. In earlier work, we isolated a cDNA for the protein prohibitin based on its preferential expression in nondividing (versus regenerating) liver and the ability of its cognate mRNA to block normal fibroblasts from entering S phase. Prohibitin was shown to be expressed in a wide range of cells; in fibroblasts, it is expressed at a high level in Gl and a low level in S. We have now cloned a cDNA for prohibition that encodes the entire protein. Prohibitin is 30 kilodaltons in size with two major domains of secondary structure. The existence of these two domains seems related to the location of prohibitin in the cell, which can be either nuclear or cytoplasmic. Prohibitin genes are highly conserved in evolution, with 75% amino acid identity between the proteins of human and fruit fly. It is therefore a strong possibility that all organisms have a prohibitin equivalent. Prohibitin is a potent negative regulator of cell growth; it can block proliferation in HeLa (cancer) cells.